Interpretive Summary: Prion diseases are the result of abnormal folding of the normal prion protein. It has been suggested that metal ions may alter the normal processing of the prion protein. This study reports increases in the normal prion protein following manganese treatment. Analysis of the mechanism of increased prion protein levels by manganese treatment indicates that the increases most likely arise due to increased resistance of the prion protein to degradation in the cell. Further analysis indicates that for this increased resistance to degradation to occur, manganese must be added to growing cells and that addition of manganese following harvest of the cells has no affect on the degradation of prion protein. These results indicate that while manganese increases prion protein levels, by increasing the resistance of prion protein to degradation, it does so in an indirect manner not through direct interaction with the prion protein.

Technical Abstract:
Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP**C) into an abnormal form of scrapie prion (PrP**Sc). The cellular mechanisms underlying the misfolding of PrP**C are not well understood. Since cellular prion proteins harbor divalent metal binding sites in the N-terminal region, we examined the effect of manganese on PrP**C processing in in vitro models of prion disease. Exposure to manganese significantly increased PrP**C levels both in cytosolic and membrane-rich fractions in a time-dependent manner. Manganese-induced PrP**C upregulation was independent of mRNA transcription or stability. Additionally, manganese treatment did not alter the PrP**C degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrP**C turnover rate was significantly altered with manganese treatment, indicating increased stability of PrP**C with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrP**C. Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse RML-infected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis.